Chlamydia trachomatis is a leading cause of sexually transmitted diseases. Understanding how this pathogen invades and replicates in host cells is fundamental to our efforts to design effective therapies that can aid in the prevention of Chlamydia infections. It has recently been shown that Chlamydia possesses genes for a type three secretion (TTS) system. In other bacteria TTS systems are a powerful virulence component shared by pathogens that allow them to subvert and invade the host. TTS systems are composed of several proteins that interact in a highly regulated manner. Chlamydia does not have a classic genetic system with which to study this complex system, thereby making this a challenging task. However, a common element to all TTS systems are the TTS chaperones which serve as a lynchpin interacting with other TTS key elements, namely, structural, effectors and regulatory proteins. We plan to exploit TTS chaperones in order to identify and characterize other key chlamydial TTS proteins. Once identified the role these proteins play, i.e. effectors, translocon or transcriptional regulator, will be established. Specifically in Aim 1 we will use TTS chaperones as bait in pull down assays and in a bacterial two-hybrid system, using targeted and genomic-wide cloning. To further characterize the proteins identified, in Aim 2 we will establish the temporal relationship of the proteins to the Chlamydia developmental cycle. Proteins will be characterized as to time of appearance and location throughout the developmental cycle, using RT-PCR, light and immuno-electron microscopy. We will also establish if proteins identified have a characteristic property of a TTS effector molecule by determining if they can be secreted by a heterologous TTS system. In other pathogens, proteins interacting with TTS chaperones have been shown to play a role in transcriptional regulation of TTS. However, nothing is known of the regulation of the Chlamydia TTS so we will first have to establish the transcriptional organization in order to determine whether the proteins identified have a role in the transcriptional regulation of the Chlamydia TTS. This will be accomplished in Aim 3 by: mapping promoters and determining the form of RNA polymerase responsible for transcription; identifying transcriptional regulators by binding to predicted regulatory elements; finally, testing the proteins identified in Aim 1 for their potential to function as a transcriptional regulator. Accomplishing these goals will aid in finding and putting the pieces of the TTS puzzle together to determine how this system functions in Chlamydia. Preventing and controlling Chlamydia infections is of great public health concern. Having an understanding of the critical elements of the Chlamydia TTS system should lead to the development of strategies to subvert the TTS system with the ultimate goal of attenuating or preventing infections with this pathogen.